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Research Article

Received: 8 February 2009

Revised: 29 April 2009

Accepted: 5 May 2009

Published online in Wiley Interscience: 11 June 2009

(www.interscience.wiley.com) DOI 10.1002/jctb.2233

Cichoric acid production from hairy root


cultures of Echinacea purpurea grown in
a modified airlift bioreactor
Bilal Haider Abbasi,a,c Rui Liu,a,d Praveen K. Saxenab and Chun-Zhao Liua,d
Abstract
BACKGROUND: Hairy root cultures of Echinacea offer great potential for the production of valuable cichoric acid, but scale-up
of the culture in the bioreactor represents a big challenge. Therefore, there is great interest in developing a suitable bioreactor
for hairy root culture of Echinacea and novel bioprocessing strategies for intensifying cichoric acid production.
RESULTS: Homogenous distribution of inoculum roots and high cichoric acid production were observed in a bioreactor modified
by installing a mesh draught tube with an average pore size 700 m, slightly larger than the hairy root, about 500 m. Improved
root growth and cichoric acid production were improved by increasing the aeration rate from 0.002 m3 h1 to 0.012 m3 h1 .
The hairy root cultures in the modified bioreactor exposed once to 6 min of ultrasound treatment at day 20 gave the highest
biomass accumulation of 12.8 0.3 g L1 , which resulted in the maximum cichoric acid production of 178.2 4.9 mg L1 at
day 30.
CONCLUSION: The present work demonstrated the effectiveness of hairy root culture in a modified airlift bioreactor. The biomass
distribution remained homogenous in the modified airlift bioreactor, and the cichoric acid production was improved owing to
the even root growth at optimal air flow rate. An interesting finding of this investigation was that ultrasound stimulated root
growth and cichoric acid production considerably in the modified airlift bioreactor.
c 2009 Society of Chemical Industry

Keywords: airlift bioreactor; cichoric acid; Echinacea purpurea; hairy roots; ultrasound

INTRODUCTION

J Chem Technol Biotechnol 2009; 84: 16971701

homogeneous root distribution as a critical parameter in the root


culture bioreactor design. However, high fluid flow resistance and
poor oxygen mass transfer still remain a big challenge for scale-up
of root culture bioreactor as hairy roots grow into a high-density
root matrix.9
Echinacea purpurea is the most common herbal medicine due to
the presence of diverse biologically active caffeic acid derivatives,
especially cichoric acid. Cichoric acid has shown phagocytic,
antihyaluronidase, antiviral activity and inhibited HIV-1 integrase
and replication.10 Commercial production of Echinacea has been
limited by a range of issues including contamination of plant
materials by microorganisms, pollution from the environment,

Correspondence to: Chun-Zhao Liu, National Key Laboratory of Biochemical


Engineering, Institute of Process Engineering, Chinese Academy of Sciences,
Beijing 100190, PR China. E-mail: czliu@home.ipe.ac.cn

a National Key Laboratory of Biochemical Engineering, Institute of Process


Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China
b Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada
N1G 2W1
c Department of Biotechnology, Quaid-i-Azam University, Islamabad-45320,
Pakistan
d Graduate School of the Chinese Academy of Sciences, Beijing 100049, P. R.
China

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Plant cell culture has been viewed as a promising alternative


to whole plant extraction for obtaining valuable chemicals.1 At
present, only a few plant cell culture processes are conducted
commercially for the production of bioactive compounds. The
major problems hindering the development of large-scale cultivation of plant cells include low productivity, cell line instability, and
difficulty in the process of scale up. Hairy root cultures offer great
potential for the production of valuable plant secondary metabolites. The advantages of using hairy roots are their independence
of plant growth regulators, high growth rates, and genetic and
biosynthetic stability.
Anatomical features of hairy roots including large longitudinalradial area ratio, lateral branching and plentiful hairiness are
the main reasons for entangled root matrix formation.2 These
structures make inoculation, tissue distribution, direct growth
measurements and the harvest of roots difficult and obstruct the
process of mass and energy transfer in the bioreactor culture. As
a result of the factors described above, hairy roots are difficult to
grow, and their scale-up in a bioreactor represents a big challenge.
A number of bioreactor configurations have been examined for
the growth of hairy roots, including gas-dispersed bioreactors3
and liquid-dispersed bioreactors.4 6 In order to achieve uniform
distribution and growth of hairy roots, some internal supports, such
as glass beads, polyurethane foam and stainless steel mesh, have
been used.4,7,8 These reports have emphasized the importance of

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variability of active components and lack of pure, standardized
plant material for biochemical analysis.11 To address these issues,
Echinacea hairy root cultures have been considered a promising
source of physiologically consistent plant tissues for a more
standardized production of the valuable cichoric acid.12 The basic
objective of this work was to investigate the performance of an
airlift bioreactor for cultivation and cichoric acid production by
hairy roots of E. purpurea. A concentric vertical stainless steel
mesh tube was employed to provide support and facilitate
distribution of the inoculated roots. In addition, the effects
of pore size of the mesh draught tube and aeration rate on
growth and cichoric acid biosynthesis in E. purpurea hairy roots
were investigated. The optimized bioreactor system was used
to assess the efficacy of ultrasound stimulation for enhancing
root growth and cichoric acid production in E. purpurea hairy
roots.

MATERIALS AND METHODS

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Hairy root cultures


The E. purpurea hairy roots were initiated and maintained as
described previously.12 Fresh mass (4 g) of the hairy roots was
subcultured every 21 days in 200 mL liquid MS medium13 with
30 g L1 sucrose in 500 mL Erlenmeyer flasks incubated at 251 C
in the dark on a rotary shaker (Model-P270, Wuhan, China) set at
100 rpm. The medium was adjusted to pH 5.8 before autoclaving
at 121 C for 20 min.

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Bioreactor system and operation


A modified airlift bioreactor for E. purpurea hairy roots was
constructed from a flanged glass column (diameter = 10 cm,
height = 25 cm) which contained a vertical stainless steel mesh
cylinder (diameter = 5 cm, height = 15.5 cm) as the draft tube for
attachment of the inoculum roots and an air sparger (compressed
stainless steel particles) underneath the bioreactor for air supply
(Fig. 1). Inside the mesh cylinder is the up-riser, and outside of
the mesh cylinder is the down-comer for mixing in the airlift
bioreactor. The working volume of the bioreactor, defined as the
volume between the top of the mesh and the bottom of the
reactor, was 1.7 L. Humidified air passed through a 0.22 m filter
(Millipore, MA) before entering the reactor through the air sparger.
Before starting the culture, the bioreactor and the air supply system
were sterilized by autoclaving at 121 C for 40 min. 34 g hairy roots
from 21-day-old shake flask cultures and 1.7 L sterilized MS liquid
medium with 30 g L1 sucrose were added into the bioreactor
system.
Stainless steel mesh with various pore sizes (380, 700, 1850, 3350
and 6700 m) were employed to determine the performance of
root growth and cichoric acid production in the modified airlift
bioreactor. After a stainless steel mesh tube with suitable pore size
was selected, different aeration rates (0.002, 0.004, 0.008, 0.012
and 0.016 m3 h1 ) were investigated and optimized for cultivating
hairy roots. An ultrasonic cleaning bath (27 22 14 cm, Model
KQ 5200DB, Shumei, China) with a fixed capacity of 40 kHz and
variable power levels was used to insonate the hairy root cultures in
the modified airlift bioreactor. The bioreactor culture was exposed
to ultrasound only once for different exposure periods of 0, 2, 4, 6
and 8 min on day 20 at log growth phase and was returned to the
normal culture condition afterward. For the ultrasound exposure,
the bioreactors containing E. purpurea hairy roots were partially
immersed in the sonic bath (at a power of 200 W) containing 8

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BH Abbasi et al.

Figure 1. Diagram of a modified airlift bioreactor equipped with a vertical


stainless steel mesh draught tube and placed inside ultrasonic bath. 1: air
outlet; 2: level of liquid medium; 3: vertical stainless steel mesh draught
tube; 4: glass column; 5: air sparger; 6: air filter; 7: airflow meter; 8 air pump;
9: electrical outlet; 10: ultrasonic bath.

L water to a depth at which the air-sparger at the bottom of the


bioreactor was about 1.0 cm below the liquid in the bath. The
bath temperature was maintained at 25 1 C with the built-in
temperature controller and by cold water. During the ultrasound
treatment, the bioreactor was left to stand vertically in water
without touching the boundary wall of the ultrasonic cleaning
bath (Fig. 1).
All bioreactor experiments were conducted at 25 1 C
in continuous light (60 mol m2 s1 ). There were triplicate
bioreactors for each treatment, and the root cultures were
harvested at day 30. Mesh draft tubes were taken out, and root
beds were easily cut using a long sharp knife.

Analytical procedures
For fresh weight (FW) determination, the hairy root cultures were
gently pressed on filter paper to remove excess water and weighed.
Subsequently, the roots were dried in an oven at 60 C for 24 h
and dry weight (DW) was recorded. Cichoric acid was estimated
according to the high performance liquid chromatography (HPLC)
analytical method described by Liu et al.,12 and anthocyanins and
lignin were analyzed according to the methods of Harborne14 and
Goering and Soest,15 respectively. Root viability was estimated by
reduction of 2, 3, 5-triphenyltetrazolium chloride (TTC) method.16
The phenylalanine ammonia lyase (PAL) activity was determined
according to the method described by Koukal and Conn,17 with
one unit of activity (U) corresponding to an absorbance variation
of 0.01.

Statistical analysis
Each of the treatments was tested three times and the data were
collected after day 30. All data presented are the mean standard
deviation (SD).

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J Chem Technol Biotechnol 2009; 84: 16971701

Cichoric acid production from hairy root cultures of E. purpurea

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RESULTS AND DISCUSSION

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120

80

40

380

700

1850

3350

6700

Dry weight (g L-1)

Dry weight
Cichoric acid
Cichoric acid (mg L-1)

Effect of pore sizes of stainless steel mesh draught tube


on growth and cichoric acid production
A modified airlift bioreactor with a concentric vertical mesh
draught tube was proposed for E. purpurea hairy root culture
for the production of cichoric acid. Pore size of the mesh draft
tube was found to be an important factor affecting growth and
cichoric acid production of E. purpurea hairy roots in the modified
airlift bioreactor. After inoculation, the inoculum roots (2.0 cm
long) remained suspended in the liquid medium following the
liquid flow pattern driven by external air supply (an aeration rate
of 0.002 m3 h1 for the first 3 days, and 0.004 m3 h1 until root
harvest at day 30), and became progressively trapped into the
mesh draught tube (Fig. 2). Among all mesh draught tubes tested,
the optimum homogeneity of inoculum roots was observed in
the modified bioreactor with mesh draught tube with an average
pore size of 700 m, compared with the hairy root with an average
diameter of about 500 m. The hairy root cultures grew evenly
along the draught tube in the modified airlift bioreactor, and
completely filled the bioreactor after 30 days. As a result, the mesh
draught tube with pore size 700 m gave the highest dry weight
of 7.8 0.2 g L1 and the maximum cichoric acid production of
102.5 5.3 mg L1 after 30 days (Fig. 3).
Bioreactors equipped with a vertical mesh cylinder for inoculum
support were reported to be favorable to hairy root growth
of Solanum chrysotrichum2 and carrot.9 Our results provide the

Pore size of mesh draught tube (um)


Figure 3. Effect of pore sizes of stainless steel mesh draught tube on growth
and cichoric acid production of E. purpurea hairy roots in the modified airlift
bioreactor. Values are means of triplicates with the standard deviation.

evidence that it is necessary to install a concentric mesh draught


tube in the airlift bioreactor in order to avoid spatial heterogeneity
developed in the culture vessel. Furthermore, it is also apparent
that the pore size of the vertical mesh draught tube plays a key
role in homogenous distribution of the initial root inoculum and
its subsequent growth.
Effect of aeration rate on growth and cichoric acid production
of E. purpurea hairy roots in the modified airlift bioreactor
In pneumatically agitated bioreactors, the volumetric gas flow rate
is a particularly important parameter affecting the rates of oxygen
transfer and broth recirculation, as well as the degree of turbulence.
The modified airlift bioreactors were operated at different rates
of aeration (0.002, 0.004, 0.008, 0.012 and 0.016 m3 h1 ) after
the first 3 days at an aeration rate of 0.002 m3 h1 . The results
presented in Fig. 4 show that the biomass increased from
5.9 0.2 g L1 to 9.0 0.3 g L1 and cichoric acid production
increased from 74.3 3.2 mg L1 to 146.5 4.5 mg L1 when the
aeration rate was increased from 0.002 m3 h1 to 0.012 m3 h1 .
No additional increments were observed in root growth or cichoric

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0
0.00

0.04

0.08

0.12

0.16

Dry weight (g L-1)

Cichoric acid (mg L-1)

Dry weight
Cichoric acid

Air flow rate (m3 h-1)

J Chem Technol Biotechnol 2009; 84: 16971701

Figure 4. Effect of aeration rate on growth and cichoric acid production of


E. purpurea hairy roots in the modified airlift bioreactor. Values are means
of triplicates with the standard deviation.

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Figure 2. Homogenous attachment of Echinacea hairy roots to the mesh


draft tube in a modified airlift bioreactor.

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Effect of ultrasound exposure on E. purpurea hairy root culture


in the modified airlift bioreactor
The stimulatory effects of ultrasound exposure on secondary
metabolism have been highlighted in shake flask cultures of plant
suspensions.20 22 In addition, the use of a low-energy ultrasoundassisted bioreactor showed improved biological activity through
enhancing mass transfer rate of gas and liquid nutrients in
microbial fermentation.23 The application of ultrasound has the
potential to enhance the growth and metabolism of Echinacea,
but this aspect has so far remained uninvestigated for Echinacea
hairy root growth and cichoric acid production in bioreactors.
To examine the effect of ultrasound exposure on the root growth
and cichoric acid production in the modified airlift bioreactor, the
E. purpurea hairy roots were exposed once to ultrasound on day
20 post-inoculation at a fixed ultrasound power for different time
periods (0, 2, 4, 6 and 8 min). As shown in Fig. 5, all sonicated root
cultures achieved higher biomass than that of control cultures
without ultrasound treatment. Of various ultrasound exposure
periods tested, a 6 min exposure at day 20 gave the highest
biomass accumulation of 12.8 0.3 g L1 , which resulted in the
maximum cichoric acid production of 178.24.9 mg L1 at day 30
The hairy root cultures treated with 6 min of ultrasound became
more purple than the control without ultrasound treatment. The
onset of purple color is related to anthocyanin accumulation and
has been shown to exist mainly in the flower of wild E. purpurea
plants.24 As shown in Fig. 6, a 6 min ultrasound treatment
enhanced anthocyanin accumulation (Fig. 6(A)) indicated by the
20

Dry weight
Cichoric acid

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120

12

80

40

4
2
6
8
Ultrasound exposure time (min)

Dry weight (g L-1)

Cichoric acid (mg L-1)

200

1700

Figure 5. Effect of ultrasound exposure on growth and cichoric acid


production of E. purpurea hairy roots in the modified airlift bioreactor.
Values are means of triplicates with the standard deviation.

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0.5
Ultrasound exposure time
6 min

0.4

8 min

Absorbance

4 min
0.3

2 min
0 min

0.2

0.1

0.0
460

6.0

Lignin content (%, w/w)

acid production with aeration rate beyond 0.12 m3 h1 . Specific


requirements of aeration for optimum growth and biochemical
output have been observed for hairy root cultures of other
medicinal species.18,19
Gasliquid oxygen transfer and liquid recirculation in the airlift
bioreactor increased with the increase of aeration rate in the
absence of root or at a low-density of root biomass. As growth
of the hairy roots proceeded, the fibrous hairy roots interlocked
and formed a tangled and porous dense matrix. As a result, the
fluid motion within the root bed became poor, especially at high
biomass densities, which possibly resulted in some limitations of
root growth and phytochemical biosynthesis due to the lack of
dissolved oxygen and nutrient supply.5

BH Abbasi et al.

4.5

480

500

520
540
Wavelength

560

580

600

3.0

1.5

0.0

2
6
4
Ultrasound exposure time (min)

Figure 6. Effect of ultrasound exposure on anthocyanin (A) and lignin


(B) accumulation in E. purpurea hairy roots in the modified airlift bioreactor.
Values are means of triplicates with the standard deviation.

wavelength maximum between 510 and 540 nm25 and lignin


content (Fig. 6(B)) in the hairy root cultures, which protected
the plant tissue from the physical stress. As shown in Fig. 7, the
increased accumulation of anthocyanins and lignin correlated well
with the increase of ultrasound-stimulated activity of PAL, a key
enzyme linked to the biosynthesis of anthocynins, caffeic acid
derivatives and lignin in plant cells.26 Exposure to ultrasound for
more than 6 min resulted in destruction of the root cells. As a result,
accumulation of cicoric acid, anthocynin and lignin declined in the
hairy root cultures.
During the ultrasound treatment, many small air bubbles
released from the dense root bed were observed in the modified
airlift bioreactor, along with an increase of root cell viability after
the ultrasound treatment (Fig. 7). The pulsation of microbubbles
of gas in the fluid generates microstreaming and other effects that
might thin the fluid boundary layer around hairy roots positioned
close to the bubbles, thus enhancing mass transfer of oxygen and
nutrient transfer from the liquid medium to the hairy roots and
removal of carbon dioxide within the high-density root matrix.27
Suitably controlled ultrasonication has shown beneficial effects in

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J Chem Technol Biotechnol 2009; 84: 16971701

Cichoric acid production from hairy root cultures of E. purpurea


10

800

Viability
PAL activity

600

400

200

Viability of roots

PAL activity (U g-1-FW)

1000

Ultrasound exposure time (min)


Figure 7. Effect of ultrasound exposure on PAL activity and viability of
E. purpurea hairy roots in the modified airlift bioreactor. Values are means
of triplicates with the standard deviation.

biological systems and biotechnological processes. These effects


appear to have multiple mechanisms that remain to be clarified.

CONCLUSIONS
The present work demonstrated the effectiveness for hairy root
culture of a modified airlift bioreactor equipped with a vertical
mesh inoculum support, which has a suitable pore size for hairy
root entrapment. The biomass distribution remained homogenous
in the modified airlift bioreactor, and cichoric acid production
was improved due to the even root growth at an optimal air
flow rate. The interesting finding of this investigation was that
ultrasound stimulated root growth and cichoric acid production in
the modified airlift bioreactor. Together, these results present new
opportunities and challenges for understanding the mechanisms
of multiple function of ultrasound on biological systems and
associated biotechnological processes involved in developing
novel bioprocessing strategies and the sonobioreactors.

ACKNOWLEDGEMENT
This work is financially supported by National Basic Research
Program (973 Program) of China (No.2007CB714301).

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